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At least 397 records · Page 22Linked to original sources
PubMed2003

Rapid detection of the known SNPs of CYP2C9 using oligonucleotide microarray.

AIM: Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. Individuals with mutant enzymes may risk serious side effects under routine therapy with certain drugs metabolized by CYP2C9. In order to facilitate the detection of the known SNPs of CYP2C9, an allele-specific oligonucleotide (ASO) based microarray was made. METHODS: An oligonucleotide microarray was made to facilitate the SNP (single nucleotide polymorphism) screening and was applied for the detection of CYP2C9 polymorphism in 62 high blood pressure (HBP) patients who received Irbesartan for treatment. Part of the genotyping results was confirmed by direct sequencing. And the relation between CYP2C9 polymorphism and therapeutic outcome of Irbesartan was statistically analyzed. RESULTS: Heterozygous alleles of CYP2C9*1/*3 were found in 7 out of 62 subjects. No mutant alleles of CYP2C9*2, *4 and *5 and no homozygous mutant alleles were detected. The 7 heterozygous CYP2C9*1/*3 and 13 random wild type DNA samples were subjected to direct sequencing with purified PCR products and same genotyping results were obtained with the 20 DNA samples. There was no significant difference in the odds of effectiveness of Irbesartan between the wild type (normal) group and CYP2C9*1/*3 (mutant) group (P>0.05). CONCLUSION: The oligonucleotide microarray made in this study is a reliable assay for detecting the CYP2C9 known alleles and the heterozygous CYP2C9*1/*3 has no significant effects on the therapeutic outcome of Irbesartan.

Alleles↗
PubMed2001

Identification of SNPs in human gamma aminobutyric acid A receptor gamma2 gene.

Gamma aminobutyric acid (GABA) is a major inhibitory neurotransmitter. Because of its importance and diverse functions, studies of single nucleotide polymorphisms in the GABA receptor genes are of great significance, which may produce useful molecular markers for genetic analysis of neurological disease, and possibly lead to the discovery of pathogenic mutations. We identified eleven SNPs in the entire exon regions, immediate intron regions and promoter region of human GABA receptor gamma2 gene by direct sequencing. In this discovery, 2 non-synonymous cSNPs and 1 splicing variant are found.

Evolution, Molecular↗
PubMed2001

[Single nucleotide polymorphisms(SNPs)and SNP databases].

Along the rapid development of human genome sequencing project, the variation data of human DNA sequence has become more and more useful not only for studying the origin, evolution and the mechanisms of maintenance of genetic variability in human populations, but also for detection of genetic association in complex disease such as diabetes, obesity, hypertension, Alzheimer's disease, etc. In recent two years, the databases such as dbSNP, CGAP, HGBASE, JST and Go!Poly etc. to collect and exploit data of genomic polymorphisms mainly single nucleotide polymorphisms (SNPs) have been respectively established in the United States, European countries, Japan and China. This overview summarized the development and applications of those SNP databases and also discussed some issues regarding the potential improvement of accuracy of SNP data collected. China has one fifth population in the world. Therefore, development of the SNP database for Chinese populations is of importance in developing complete SNP databases of human genome and may also stimulate the further development of biomedical research and production in China.

Databases, Nucleic Acid↗
PubMed2002

Primer design for PCR and sequencing in high-throughput analysis of SNPs.

To achieve high-throughput analysis of allele frequencies in human SNPs, we have developed automated methodsfor designing PCR and DNA sequencing primers. We found we could run the PCR assays at quite stringent, uniform conditions. The design process used freely available databases, including dbSNP, SNPper, and TSC, and publicly available software including RepeatMasker and Primer3. We describe parameters for the software and other considerations that increase experimental success. As anticipated. some assays filed at the design stage due primarily to the genomic locations of repetitive sequences, extreme GC content regions, or lack of sufficient sequence. However, over 23,000 assays, including 96% of those recently analyzed, have been experimentally successfuL Similar design methods could be usedfor PCR assays in any organism with substantial available sequence.

DNA Mutational Analysis↗
PubMed2005

Searching for candidate genes in acute lung injury: SNPs, Chips and PBEF.

Acute lung injury (ALI) is a devastating illness, occurring in the setting of sepsis, with genetic variations contributing to ALI susceptibility and severity. We utilized the "candidate gene approach" with extensive expression profiling in animal and human ALI models to identify novel candidate genes. We noted significant expression of pre-B-cell colony enhancing factor (PBEF), a gene not previously associated with lung pathophysiology. This finding was validated by molecular, biochemical and immunohistochemical approaches with increased levels of PBEF also detected in human BAL and serum. DNA sequencing identified two single nucleotide polymorphisms (SNPs) in the PBEF promoter (T-1001G, C-1543T), which were genotyped in a Caucasian cohort of sepsis-associated ALI patients. Carriers of the GC haplotype exhibited a 5.7-fold relative ALI risk compared to controls associated with increased PBEF promoter activity. These studies demonstrate the successful application of genomic technologies in the identification of novel candidate genes in complex lung disease.

Alleles↗
PubMed2006

[Analysis of SNPs in exons 2 and 3 of the leptin gene in wild boar, swine and hybrid swine].

Leptin is a 167-amino acid hormone mainly expressed in the fat tissue. It serves as an important regulator of energy uptake and consumption through action on the hypothalamus. Herein single nucleotide polymorphisms (SNPs) were detected in exons 2 and 3 of the Leptin gene in wild boar, swine and half-bred swine by PCR-SSCP. Cloning and sequencing of homozygous fragments revealed the following variations in the coding region: a silent T to C transversion at nucleotide 214; CG to GC at nucleotides 364 and 365, which converts Arg to Ala; a silent G to A transversion at nucleotide 426; a T insertion at nucleotide 451 resulting in frameshift; and a G to T transversion at nucleotide 462.

Amino Acid Substitution↗
PubMed2006

Surveyor nuclease-based genotyping of SNPs.

Surveyor nuclease, a single-strand-specific endonuclease that cleaves DNA at the 3' side of a base-mismatch site in both DNA strands, can be used for genotyping of SNPs by preparing two different types of a heteroduplex: one from mutant DNA fragments and the other from a mixture of wild-type and mutant fragments. We show an example of this technique and propose that it can be used with some advantages instead of RFLP analysis.

DNA↗
PubMed2006

Genetic polymorphisms of human ABC transporter ABCG2: development of the standard method for functional validation of SNPs by using the Flp recombinase system.

The vector-mediated introduction of cDNA into mammalian cells by calcium phosphate co-precipitation or permeation with lipofectamine is widely used for the integration of cDNA into genomic DNA. However, integration of cDNA into the host's chromosomal DNA occurs randomly at unpredictable sites, and the number of integrated recombinant DNAs is not controllable. To investigate the effect of genetic polymorphisms of ABCG2 on the protein expression and the drug resistance profile, we developed the Flp-In method to integrate one single copy of ABCG2 variant-cDNA into FRT-tagged genomic DNA. More than 20 metaphase spreads were examined for both fluorescence in situ hybridization (FISH) mapping and multicolor-FISH analysis, and it has been revealed that ABCG2 cDNA was incorporated into the telomeric region of the short arm on one of chromosomes 12 in Flp-In-293 cells. Based on the currently available SNP data for human ABCG2, we have created a total of seven variants by site-directed mutagenesis and stably expressed them in Flp-In-293 cells. While mRNAs of those integrated ABCG2 variants and wild type were evenly expressed in Flp-In-293 cells, the protein expression levels of F208S and S441N variants were found to be markedly low. It is suggested that the protein instability due to enhanced degradation resulted in the low levels of their protein expression. Thus, the Flp recombinase system would provide a useful tool to validate the effect of nonsynonymous SNPs on the protein stability and post-translational modification of ABCG2.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2002

Evaluation of multiple presenilin 2 SNPs for association with early-onset sporadic Alzheimer disease.

The presenilin genes encode proteins that modify, mediate, or perform similar functions to gamma-secretase, the enzyme responsible for converting amyloid beta precursor protein (APP) into beta-amyloid. Mutations in the presenilin genes cause an increased production of Abeta42, the aberrant form of beta-amyloid found in the neural plaques of Alzheimer disease patients. Previously reported association studies of presenilin 2 (PSEN2) polymorphisms with early-onset Alzheimer disease (EOAD) have produced contradictory results. In an effort to resolve these differences, we tested eight single nucleotide polymorphisms in and around the 3' region of the PSEN2 gene for association with EOAD. An initial set of Scottish EOAD cases (n = 121) and controls (n = 152) was screened using the genotyping method dynamic allele-specific hybridization (DASH). No significant differences were seen between allele or genotype frequencies of cases and controls. However, when conditioned on the risk allele (epsilon 4) APOE, three polymorphisms showed allelic association with a P value below 0.05. These same polymorphisms were in near 100% linkage disequilibrium with each other (P < 5 x 10(-5)), and in each, one of the homozygous genotypes was absent in controls but present in the cases. Replication in an independent set of Scottish EOAD cases (n = 84) and controls (n = 173) did not confirm this finding. From this study we find no evidence to suggest that variations in the PSEN2 gene pose as major risk factors for sporadic EOAD.

3' Untranslated Regions↗
PubMed2006

Genotyping fetal paternally inherited SNPs by MALDI-TOF MS using cell-free fetal DNA in maternal plasma: influence of size fractionation.

The determination of fetal point mutations from fetal cell-free DNA (cf-DNA) in maternal plasma is technically challenging due to the preponderance of maternal sequences. It has recently been shown that fetal cf-DNA sequences are smaller than maternal ones and that the selection of small cf-DNA fragments by size fractionation by agarose gel electrophoresis leads to the enrichment of fetal cf-DNA sequences, thereby permitting the detection of otherwise masked fetal point mutations. In a separate development, the use of MALDI-TOF MS has also been shown to facilitate the detection of fetal point mutations from cf-DNA in maternal plasma. In this study, a combination of these approaches was examined. cf-DNA was extracted from 18 maternal plasma samples, 10 taken at term and 8 obtained early in the second trimester. A total of 41 SNP loci were examined in size-fractionated and total cf-DNA using either a conventional homogeneous MassEXTEND (hME) assay or a nucleotide-specific single allele base extension reaction (SABER) assay. The analysis of total cf-DNA indicated that size fractionation considerably enhanced the sensitivity of the standard hME assay, especially for samples taken early in pregnancy. Size fractionation also rendered the signals obtained by the SABER assay more precise.

Alleles↗
PubMed2006

Estimating haplotype relative risks in complex disease from unphased SNPs data in families using a likelihood adjusted for ascertainment.

The understanding of complex diseases and insights to improve their medical management may be achieved through the deduction of how specific haplotypes may play a joint effect to change relative risk information. In this paper we describe an ascertainment adjusted likelihood-based method to estimate haplotype relative risks using pooled family data coming from association and/or linkage studies that were used to identify specific haplotypes. Haplotype-based analysis tends to require a large amount of parameters to capture all the information that leads to efficiency problems. An adaptation of the Stochastic Expectation Maximization algorithm is used for haplotypes inference from genotypic data and to reduce the number of nuisance parameters for risk estimation. Using different simulations, we show that this method provides unbiased relative risk estimates even in case of departure from Hardy-Weinberg equilibrium.

Algorithms↗
PubMed2004

Proofreading genotyping assays and electrochemical detection of SNPs.

The use of proofreading DNA polymerases in genotyping assays offers the prospect of improved performance. To this end, we have recently used compatible DNA polymerases, protected primers, and substrates to implement proofreading single base extension (P-SBE) and proofreading allele-specific extension (PRASE) assays. Key aspects of the P-SBE and related proofreading assay formats are described here. For transduction of genotyping reactions into physical signals, electrochemical SBE implementations may offer simple, inexpensive assays in electrode array or electrophoretic formats. We have developed electrochemically-labeled nucleotides and electrode detection methods with a view to these applications. Detection of electrochemically-labeled SBE products on an oligonucleotide-modified gold electrode surface is demonstrated.

DNA Mutational Analysis↗
PubMed2002

Quantitative technologies for allele frequency estimation of SNPs in DNA pools.

We have compared several genotyping methods to assess their applicability to single nucleotide polymorphism (SNP) allele frequency estimation in DNA pools. The accuracy of these methods (restriction fragment length polymorphism, real-time pyrophosphate DNA sequencing, single base extension with fluorescently labeled ddNTPs, homogeneous 5'-nuclease assay, and MALDI-TOF mass spectrometry) was tested by calculating the standard deviation among heterozygous individuals (which are natural DNA pools with 50% representation of each allele) and by estimating allele frequency in artificial pools. We show that although the methods differ in their accuracy, they can all serve for quantification of allele frequency in DNA pools with reasonable accuracy. We found that the influence of the error variance attributed to pool construction on quantification accuracy is insignificant and is SNP dependent.

Algorithms↗
PubMed2004

Identification of SNPs in the waxy gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice.

Common non-waxy ( Wx) rice cultivars contain two different alleles at the waxy locus, designated Wx(a) and Wx(b), which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wx(a) allele was predominant in non-waxy indica cultivars, whereas the Wx(b) allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wx(a) from Wx(b) have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AG GT to AG TT) at a donor site of the first intron within the Wx gene. In the case of waxy ( wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wx(a) or Wx(b) alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wx(a) allele (AGGT) but not in the Wx(b) allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wx(a)- and Wx(b)-derived alleles, but that the Wx(b)-derived allele was predominant, and its distribution was independent of indica- japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples.

Alleles↗